Restrained multifunctional reagent for surface modification

ABSTRACT

A multifunctional reagent is provided that is useful for the attachment of desired molecules to support surfaces. A reactive reagent molecule of the invention is &#34;restrained&#34; in that it is conformationally and/or chemically restricted from reacting with either itself or with other molecules of the same reagent. Upon activation, this feature causes the attachment of less than all of the reactive sites of the multifunctional reagent to a surface, thereby leaving the remaining sites free to react with molecules desired to be immobilized onto the surface.

TECHNICAL FIELD

The present invention relates to chemical and/or physical modificationof the surface properties of industrially and medically importantsubstrates. In a further aspect, the present invention relates to thevarious processes useful for modifying the surface properties of bulkmaterials for specific applications. In this aspect, the presentinvention relates to such surface modification techniques as plasmadeposition, radiation grafting, grafting by photopolymerization, ionimplantation, and chemical derivatization.

BACKGROUND OF THE INVENTION

The chemical modification of surfaces, to achieve desired chemicaland/or physical characteristics, has been previously described. U.S.Pat. Nos. 4,722,906; 4,973,493; 4,979,959; and 5,002,582 (thedisclosures of each of which are incorporated herein by reference), forexample, relate to surface modification by the use of latent reactivegroups to achieve covalent coupling of reagents such as biomolecules andsynthetic polymers to various substrates. The preferred latent reactivegroup is typically described as a photochemically reactive functionalgroup (i.e., photoreactive group) that, when exposed to an appropriateenergy source, undergoes a transformation from an inactive state (i.e.,ground state) to a reactive intermediate capable of forming covalentbonds with appropriate materials.

Such latent reactive groups are typically described as being used tofirst derivatize a desired compound (e.g., thermochemically), followedby the application (photochemically) of the derivatized compound to asurface. Such a sequential approach is suitable in many situations, butthe approach can lack such attributes as speed, versatility, and ease ofuse, such as for target molecules that are inherently difficult to firstderivatize.

What would be clearly desired would be a reactive reagent that providesan optimal combination of the speed, versatility, and ease of usenecessary for the derivatization of suitable surfaces, particularly onethat is useful either simultaneously with the application of a targetmolecule, or one that can be used to prime a surface prior to theapplication of a target molecule.

SUMMARY OF THE INVENTION

We have discovered a novel restrained, multifunctional reagent usefulfor prior derivatization of a support surface, or for simultaneousapplication with a target molecule to a support, the reagent comprisinga chemical backbone having attached to it one or more first latentreactive groups and one or more second latent reactive groups, each ofthe first and second latent reactive groups being attached to thebackbone in such a manner that, upon activation of the latent reactivegroups in the presence of a support surface,

a) the first latent reactive groups are capable of covalently bonding tothe support surface, and

b) upon bonding of the first latent reactive groups to the surface, thesecond latent reactive groups are;

i) restricted from reacting with either a spacer or the support surface,

ii) capable of reverting to their inactive state, and

iii) upon reverting to their inactive state, are thereafter capable ofbeing reactivated in order to later bind a target molecule, therebyattaching the target molecule to the surface..

In a particularly preferred embodiment, the chemical backbone of such anultifunctional reagent is a single tetrahedral carbon atom. Attached tothe central carbon, in this embodiment, are four identical latentreactive groups, in the form of photoreactive groups, each attached viaidentical spacer chains. Upon exposure to a suitable light source, eachof the latent reactive groups are subject to activation.

By virtue of conformational and/or steric constraints that the reagentimposes on itself (hence "restrained"), both by the tetrahedral natureof the central carbon, as well as the physical-chemical nature of thespacer chains themselves (e.g., their length, reactivity, andflexibility), the reagent is restricted, in that a maximum of three ofthe four activated latent reactive groups on any given preferred reagentmolecule are able to attach to the support surface. The remainingunreacted group(s) are thus able to revert to their inactive state. Onecan visualize the resultant structure as being analogous to afour-pronged child's jack being tossed onto a table. Three of the prongswill rest on the surface of the table, with the fourth pointing up andaway from the table.

In a subsequent step, the unreacted group(s) can be reactivated in thepresence of a target molecule, in order to covalently bond the targetmolecule to the surface.

The reagent of the present invention has broad applicability,particularly since it can be used to provide a "primed" surface, i.e., asurface having latent reactivity for a target molecule. The reagent istherefore particularly useful in situations where the available quantityof the target molecule is limited; where prior derivatization of atarget molecule would create an insoluble or inactive product; or wherethere is a desire to prepare and store a primed surface for later use,e.g., with a variety of target molecules.

The reagent can also be used to prepare a primed latent reactive surfacefor the subsequent application of a target molecule that has itself beenpreviously derivatized with latent reactive groups, i.e., latentreactive groups provided by compounds other than what may be present inthe respective restrained reagent. This approach could be useful forproviding increased sites of bonding between the surface and the targetmolecule.

Additionally, the reagent provides a further benefit in that it can beused in a mixture with target molecules (nonderivatized or previouslyderivatized), in the presence of a surface, to permit simultaneousapplication (in contrast to the sequential application described above)in the course of surface modification.

DETAILED DESCRIPTION

The reagent of the present invention involves a chemical backbone havingattached to it one or more first latent reactive groups capable ofattaching to a surface, and one or more second latent reactive groupscapable of attaching to a target molecule intended for immobilization.Chemically, the first and second latent reactive groups, and respectivespacers, can be the same or different.

In situations in which all latent reactive groups and spacers arechemically, or at least functionally, the same, the distinction betweenfirst and second latent reactive groups may actually be accomplished atthe time of the first activation step, i.e., those groups that areactivated and attach to the surface will be considered "first" latentreactive groups, and those that remain unreacted (whether or not theyhave been activated) will be considered "second" latent reactive groups.

The first and second latent reactive groups are preferably attached tothe backbone by spacer chains in such a manner that, upon activation ofthe latent reactive groups in the presence of a support surface, thefirst latent reactive groups are capable of covalently bonding to thesurface. The second latent reactive groups are thereby conformationallyrestricted, thus preventing reaction with either their spacers, otherrestricted reagents of the same type, or the support surface. Inaddition, after the first activation step and removal of the activatingstimulus (e.g., illumination source), the second latent reactive groupsare capable of reverting to their inactive state and can thereafter beactivated (or reactivated, as the case may be) to covalently bond atarget molecule.

The following diagram depicts the concept of the preferred tetrahedralcore structure, as exemplified by the empirical formula X(Y)₄ (Z)₄,shown below as Formula I: ##STR1## In Formula I:

    ______________________________________                                        X                = the chemical backbone;                                     Y.sub.1, Y.sub.2, Y.sub.3, Y.sub.4                                                             = optional spacers; and                                      Z.sub.1, Z.sub.2, Z.sub.3, Z.sub.4                                                             = latent reactive groups.                                    ______________________________________                                    

As used herein, the term "chemical backbone" refers to the atom, orother molecular structure, to which either the latent reactive groups orspacers are attached, and which provides, .at least in part, the desiredsteric and conformational restrictiveness between groups or spacers thatare attached to the same chemical backbone. The term "core molecule"will be used to refer to the combination of chemical backbone and anyattached spacers (i.e., "X+Y" in Formula I above), that is, withoutlatent reactive groups.

The term "latent reactive group", as described more fully below, willrefer to the activatible group attached to a spacer, that is used tobond with either the support surface ("first" latent reactive group) orthe target molecule ("second" latent reactive group). The word "active"refers to a latent reactive group that has been subjected to anappropriate stimulus, in order to render it capable of forming acovalent bond with a suitable moiety. The word "inactive" refers to alatent reactive group either before activation, or after one or morecycles of activation and reversion to the inactive state. The term"target molecule", in turn, will be used to refer to the molecule thatis intended to be attached to the surface, via the reagent, generally inorder to provide the desired characteristics conveyed by virtue of itsbinding.

In a particularly preferred embodiment, the invention provides a coremolecule containing four dimethyleneoxy groups (--CH₂ --O--CH₂ --)bonded as spacers to a central tetrahedral carbon atom, the carbon atomserving in this instance as the chemical backbone. The backbone,spacers, and latent reactive groups are described herein, for the sakeof simplicity, as being distinct portions of the reagent of the presentinvention. In the chemical synthesis .of a reagent however, theseportions will rarely be provided as three independent precursors.Instead, and most often, the portion referred to herein as the spacerwill be formed as the result of the reaction between two molecules, onethat contains the core molecule and another that contains the latentreactive group.

By virtue of the physical and chemical properties of the photoreactivegroups and the methylene group spacers, together with the conformationalrestrictions provided by the tetrahedral carbon backbone, the reagent isable to attach up to three of its photoreactive groups to a surface uponphotoactivation. Being conformationally restricted, and thus unable tointeract with the support surface or the spacers, any remainingphotoreactive group(s) are able to return to their inactive states uponremoval of fight, once again being capable of activation by subsequentillumination.

In addition to reagents of the particularly preferred embodiment,containing a central carbon atom, reagents of the present invention canbe prepared having any suitable chemical (e.g., organic and/orinorganic) backbone structure, including those that employ a singleatom, such as silicon, nitrogen, phosphorus, and any other atom withfour or more bonds nonplanar with respect to one another.

Also, molecules having conformationally restricted ring structures (suchas inositol, i.e., hexahydroxy cyclohexane) can be derivatized withlatent reactive groups in a manner analogous to that described hereinfor pentaerythritol, to provide latent reactive groups in both axial andequatorial positions. Other polyhydroxylated compounds such as mono- anddi-saccharides, and cyclodextrins, are suitable as well, in that theyoffer alternative opportunities to create other multisubstitutedreagents having varying placements and densities of latent reactivegroups.

Contact with a support surface and activation of the latent reactivegroups will result in covalent bond formation through at least onelatent reactive group, with at least one other latent reactive groupbeing conformationally restricted and thus unable to react at thesurface.

Spacers useful in the reagent of the present invention can be bonded tothe tetrahedral atom and can be of any suitable length and structure. A"spacer", as used herein, refers to that region of a reagent between alatent reactive group and a chemical backbone. The use of spacers isoptional, and would not be necessary, for instance, for such compoundsas acylated derivatives of tetraphenylmethane having the structure shownbelow as FORMULA II: ##STR2##

Functionally, it is particularly preferred that a spacer does not haveany groups or atoms that would be both physically accessible to, andchemically reactive with, an activated latent reactive group (whetherfrom the same or another reagent molecule), to an extent that wouldrender the reagent useless for its intended purpose. At the very least,the spacer should have no atom or groups that would kinetically competewith the binding of latent reactive groups to their intended target, beit a surface or a target molecule. For instance, preferred spacersshould typically not have any accessible "abstractable hydrogen" atoms,i.e., hydrogen atoms that are accessible to and reactive with theactivated latent reactive group of choice.

Molecular modeling techniques, as are available to and within the skillof those in the art, can be used to determine the optimal length andstructure of spacers needed to keep latent reactive groupsconformationally restricted from reacting. Typically the spacer willhave no linear region longer than about 5 atoms (i.e., 6 bonds), andpreferably 4 atoms (5 bonds), in length. Although it is not requiredthat the spacers within a particular reagent be chemically identical,the use of different spacers in a single reagent molecule is notgenerally preferred; in that such an embodiment will typically requiremore synthetic steps and may require more complex chemical separationsin their preparation.

Constituent atoms of the spacers need not be aligned linearly. Forexample, aromatic rings, which lack abstractable hydrogen atoms (asdefined above), can be included as part of spacer design in thosereagents where the latent reactive group functions by initiatingcovalent bond formation via hydrogen atom abstraction. In its precursorform (i.e., prior to attachment of a latent reactive group), a spacercan be terminated with any suitable functionality, such as hydroxyl,amino, carboxyl, and sulfhydryl groups, which is suitable for use inattaching a latent reactive group by a suitable chemical reaction, e.g.,conventional coupling chemistry.

A "latent reactive group", as used herein, refers to a chemical groupthat responds to an applied external energy source in order to undergoactive specie generation, resulting in covalent bonding to an adjacentchemical structure (e.g., an abstractable hydrogen). Preferred groupsare sufficiently stable to be stored under conditions in which theyretain such properties. See, e.g., U.S. Pat. No. 5,002,582, thedisclosure of which is incorporated herein by reference. Latent reactivegroups can be chosen that are responsive to various portions of theelectromagnetic spectrum, with those responsive to ultraviolet andvisible portions of the spectrum (referred to herein as "photoreactive")being particularly preferred.

Photoreactive aryl ketones such as acetophenone and benzophenone, ortheir derivatives, are preferred, since these functional groups,typically, are readily capable of undergoing theactivation/inactivation/reactivation cycle described herein.Benzophenone is a particularly preferred photoreactive group, since itis capable of photochemical excitation with the initial formation of anexcited singlet state that undergoes intersystem crossing to the tripletstate. The excited triplet state can insert into carbon-hydrogen bondsby abstraction of a hydrogen atom (from a support surface, for example),thus creating a radical pair. Subsequent collapse of the radical pairleads to formation of a new carbon-carbon bond. If a reactive bond(e.g., carbon-hydrogen) is not available for bonding, the ultravioletlight-induced excitation of the benzophenone group is reversible and themolecule returns to ground state energy level upon removal of the energysource. Hence, photoreactive aryl ketones are particularly preferred.

The method of the present invention involves the attachment of a targetmolecule to a support surface by use of the above-described reagent,. Aswill be discussed more fully below, the reagent can be used in a numberof different ways to achieve the desired result.

The method of the present invention comprises the steps of

A. Providing a multifunctional reagent comprising a chemical backbonehaving attached to it one or more first latent reactive groups and oneor more second latent reactive groups, each of the first and secondlatent reactive groups being attached to the backbone in such a mannerthat upon activation of the latent reactive groups in the presence of asupport surface,

(1) the first latent reactive groups are capable of covalently bondingto the surface, and

(2) upon bonding of the first latent reactive groups to the surface, thesecond latent reactive groups are

a. conformationally restricted from reacting with either a spacer or thesupport surface,

b. capable of reverting to their inactive state, and

c. upon reverting to their inactive state, capable of being reactivatedin order to later bind a target molecule in order to attach the targetmolecule to the surface,

B. activating the first latent reactive groups in the presence of thesupport surface, in order to bond the first latent reactive groups tothe surface, and

C. activating the second latent reactive groups in the presence of thetarget molecules, in order to bond the second latent reactive groups tothe target molecules, thereby attaching the target molecules to thesurface.

The steps of the method can be performed in arty suitable order. Forexample, a multifunctional reagent as described above can physicallyabsorb itself to a suitable support surface by hydrophobic interactions.Upon illumination, the photoreactive groups (e.g., benzophenone groups)undergo covalent bond formation at the support surface by theaforementioned mechanism. Given the conformational restrictions of thetetrahedral bonding core atom, at least one, and up to three of the fourphotoreactive groups form bonds with the surface. With the absence ofabstractable hydrogens in the proximity of the remaining unbondedphotoreactive group(s), and removal of the illumination source, theexcited state benzophenone returns to ground state energy. Theseremaining groups are then capable of being reactivated when the targetmolecule intended for immobilization is present and when the treatedsurface is exposed to another round of illumination. This method can bedescribed as a "two-step" approach, where the photoreactive reagent isapplied in the first step to create the latent reactive surface, and inthe second step, the target molecule is added for attachment to theactivated surface.

In another embodiment, which can be described as a "one-step" method,the reagent of the present invention is mixed in solution with thetarget molecule to form a binary composition, and this composition isused to surface modify materials in a single illumination step. In thiscase, illumination triggers not only covalent bond formation of thelatent reactive group with the material surface, but also simultaneouslytriggers covalent bond formation with adjacent target molecules residingon the surface. In the course of this process, however, the reagent issubstantially precluded from bonding to other reagent molecules byvirtue of conformational restrictions and/or the lack of abstractablehydrogen atoms.

In yet another embodiment, the invention provides a method of using amultifunctional reagent to pretreat a substrate surface prior to theapplication and bonding of molecules that have themselves beenfunctionalized with latent reactive groups. This method is useful insituations where a particularly difficult substrate requires maximalcoating durability. In this manner, the number of covalent bonds formedbetween the substrate surface and the target molecule derivatized withlatent reactive groups can typically be increased, as compared tosurface modification with a desired latent reactive group-containingtarget molecule alone. This approach offers significant advantages, e.g,in terms of increasing the tenacity of binding of the desired moleculeto the surface, without having to increase the latent reactive groupcontent of the target molecule to a point where properties such as thesolubility or functional activity of the molecule would be impaired.

In view of the present disclosure, reagents of the present invention canbe prepared according to conventional synthetic methods. A preferredreagent, for instance, can be prepared according to the followingprotocol: A mixture of the core molecule (e.g., pentaerythritol) and anexcess of a derivative of the latent reactive group (e.g.,4-bromomethylbenzophenone) are dissolved in a suitable solvent andrefluxed in the presence of a base capable of alkoxide anion generation.The product, a tetrakis (4-benzoylbenzyl ether) of pentaerythritol canthen be purified by preparative chromatography. The product has thestructure shown below as FORMULA III: ##STR3##

Any suitable coupling chemistry can be used to attach the latentreactive group to the core molecule. For example, an ester couplinggroup can be prepared by reaction of 4-benzoylbenzoyl chloride withpentaerythritol, using a suitable solvent and acid scavenger. Similarly,a urethane coupling group can be generated by reaction of 4-benzophenoneisocyanate with pentaerythritol. Also, where the tetrahedral coremolecule contains spacers terminated with amine functional groups, asopposed for instance to hydroxyl groups, a latent reactive group can beintroduced via an amide functionality, using an acid chloride or anN-oxysuccinimide ester.

Likewise, if the core molecule spacers are terminated with sulfhydrylgroups, a maleimide-substituted latent reactive group can be used in thecoupling reaction. The coupling reaction of the core molecule (such aspentaerythritol) with the latent reactive group can be preceded by thesynthesis of a core molecule that includes not only the pentaerythritolprecursor but also spacer extensions based on molecules that arenonreactive or sterically hindered with respect to reaction with thelatent reactive group.

Reagents of the present invention can be used to modify any suitablesurface. Where the latent reactive group of the reagent is aphotoreactive group of the preferred type, it is particularly preferredthat the surface provide abstractable hydrogen atoms suitable forcovalent bonding with the activated group.

Plastics such as polyolefins, polystyrenes, poly(methyl)methacrylates,polyacrylonitriles, poly(vinylacetates), poly (vinyl alcohols),chlorine-containing polymers such as poly(vinyl) chloride,polyoxymethylenes, polycarbonates, polyamides, polyimides,polyurethanes, phenolics, amino-epoxy resins, polyesters, cellulosederivatives, silicones, and rubber-like plastics can all be used assupports, providing surfaces that can be modified as described herein.See generally, "Plastics", pp. 462-464, in Concise Encyclopedia ofPolymer Science and Engineering, Kroschwitz, ed., John Wiley and Sons,1990, the disclosure of which is incorporated herein by reference. Inaddition, supports such as those formed of pyrolytic carbon andsilylated materials such as glass ceramic, or metal are suitable forsurface modification.

Suitable target molecules for use in the present invention, forattachment to a support surface, encompass a diverse group ofsubstances. Target molecules can be used in either an underivatizedform, or previously derivatized. Moreover, target molecules can beimmobilized singly or in combination with other types of targetmolecules. In addition, target molecules can be immobilized to thesurface either after (e.g., sequentially), or during (e.g.,simultaneously with) attachment of the present multifunctional reagentto the surface.

Typically, target molecules are selected so as to confer particulardesired properties to the surface and/or to the device or articlebearing the surface. Examples of suitable target molecules, and thesurface properties they are typically used to provide, is represented bythe following non-limiting list:

    ______________________________________                                        TARGET MOLECULE  FUNCTIONAL ACTIVITY                                          ______________________________________                                        Synthetic Polymers                                                            Sulfonic acid-substituted                                                                      Lubricity, negatively charged                                polyacrylamide   surface, hydrophilicity                                      Polyacrylamide   Lubricity, protein repulsion,                                                 hydrophilicity                                               Polyethylene glycol                                                                            Lubricity, cell and protein                                                   repulsion, hydrophilicity                                    Polyethyleneimine                                                                              Positively charged surface                                   Polylactic acid  Bioerodible surface                                          Polyvinyl alcohol                                                                              Lubricity, hydrophilicity                                    Polyvinyl pyrrolidone                                                                          Lubricity, hydrophilicity                                    Quaternary amine-substituted                                                                   Lubricity, positively charged                                polyacrylamide   surface                                                      Silicone         Lubricity, hydrophobicity                                    Conductive polymers (e.g.,                                                                     Electric conductivity                                        polyvinylpyridine,                                                            polyacetylene, polypyrrole)                                                   Carbohydrates                                                                 Alginic acid     Lubricity, hydrophilicity                                    Cellulose        Lubricity, hydrophilicity, biode-                                             gradable glucose source                                      Chitosan         Positively charged surface,                                                   hydrophilicity                                               Glycogen         Hydrophilicity, biodegradable                                                 glucose source                                               Heparin          Antithrombogenicity, hydro-                                                   philicity, cell attachment                                   Hyaluronic acid  Lubricity, negatively charged                                                 surface                                                      Pectin           Lubricity, hydrophilicity                                    Mono-, di- saccharides                                                                         Hydrophilicity                                               Dextran sulfate  Chromatography media                                         Proteins                                                                      Antibodies       Antigen binding                                              Antithrombotic agents (e.g.,                                                                   Antithrombogenic surface                                     antithrombin III)                                                             Albumin          Nonthrombogenic surface                                      Attachment proteins/peptides                                                                   Cell attachment                                              (e.g. collagen)                                                               Enzymes          Catalytic surfaces                                           Extracellular matrix proteins/                                                                 Cell attachment and growth                                   peptides                                                                      Growth factors, proteins/                                                                      Cell growth                                                  peptides                                                                      Hirudin          Antithrombogenic surface                                     Thrombolytic proteins (e.g.,                                                                   Thrombolytic activity                                        streptokinase, plasmin,                                                       urokinase)                                                                    Lipids                                                                        Fatty acids      Hydrophobicity, biocompatibility                             Mono-, di- and triglycerides                                                                   Hydrophobicity, lubricity, bio-                                               degradable fatty acid source                                 Phospholipids    Hydrophobicity, lubricity, biode-                                             gradable fatty acid source                                   Prostaglandins/leukotrienes                                                                    Nonthrombogenic surface/                                                      immobilized messengers                                       Nucleic Acids                                                                 DNA              Substrate for nucleases/                                                      affinity binding                                             RNA              Substrate for nucleases/                                                      affinity binding                                             Nucleosides, nucleotides                                                                       Source of purines, pyrimi-                                                    dines, enzyme cofactors                                      Drugs/vitamins/cofactors                                                      Enzyme cofactors Immobilized enzymes                                          Heme compounds   Globin bindings/surface oxygen-                                               ation                                                        Drugs            Drug activity                                                Non-polymeric Materials                                                       Dyes (e.g., azo dyestuffs)                                                                     Coloring agents                                              Fluorescent compounds                                                                          Fluorescence                                                 (e.g., fluorescein)                                                           ______________________________________                                    

Any suitable technique can be used for reagent attachment to a surface,and such techniques can be selected and optimized for each material,process, or device. The multifunctional reagent has been successfullyapplied to clean material surfaces as listed above by spray, dip, orbrush coating of a solution of the reactive reagent. In a typicalapplication, the support intended for coating is first dipped in asolution of the reagent diluted with a suitable solvent (e.g., isopropylalcohol). The reagent-coated support is then exposed to ultravioletfight in order to promote covalent bond formation at the materialsurface. After washing to remove any unbound reagent, application of themolecule intended for immobilization, followed by a second UVillumination, results in surface modification with the target molecule.

When desired, other approaches can be used for surface modificationusing the reagent of the present invention. For example, the latentreactive reagent can be mixed directly with the molecule intended forimmobilization. The tetrakis (4-benzoylbenzyl ether) of pentaerythritolat 0.1 mg/ml when mixed with nitrocellulose at 40 mg/ml can be used tosurface modify polyvinylidene difluoride membranes with a singleillumination step. In another experimental approach, the photoreactivederivatives of these synthetic and naturally occurring molecules canalso be applied to surfaces pretreated with multifunctional reagentssuch as the tetrakis (4-benzoylbenzyl ether) of pentaerythritol. Thisapproach is particularly useful in those situations in which a supportis difficult to modify using conventional chemistry, or for situationsthat require exceptional durability and stability of the target moleculeon the surface.

The present invention provides a reagent and method useful for alteringthe surface properties of a variety of devices of medical, scientific,and industrial importance, using a broad spectrum of suitable targetmolecules.

The invention will be further described with reference to the followingnon-limiting Examples. It will be apparent to those skilled in the artthat many changes can be made in the embodiments described withoutdeparting from the scope of the present invention. Thus the scope of thepresent invention should not be limited to the embodiments described inthis application, but only by embodiments described by the language ofthe claims and the equivalents of those embodiments.

EXAMPLES EXAMPLE 1 Preparation of Tetrakis (4-benzoylbenzyl ether) ofPentaerythritol ["tetra-BBE-PET"]

Pentaerythritol [Aldrich] (2.0 g; 14.71 mmole; dried at 60° C. at <1 mmHg for 1 hour), 4-bromomethylbenzophenone (20.0 g; 72.7 mmole; preparedby free radical bromination of 4-methylbenzophenone [Aldrich]), 80%(w/w) sodium hydride in mineral oil [Aldrich] (NaH 1.23 g; 41.0 mmole),and tetrahydrofuran ("THF", 120 ml) were refluxed for 34 hours in anargon atmosphere. An additional amount of 80% NaH (2.95 g; 98.3 mmole)was then added to the reaction mixture, and the mixture refluxed for anadditional 7 hours under argon. The reaction was quenched by theaddition of 8 ml of glacial acetic acid (HOAc). The quenched reactionwas centrifuged to aid in the removal of THF insolubles.

The liquid was decanted, and the insolubles were washed with three 50 mlportions of chloroform (CHCl₃). The decanted liquid (mainly THF) and theCHCl₃ washes were combined and evaporated to give 18.7 g of a crudeyellow semi-solid residue. A portion of the crude product (2 g) waspurified by flash chromatography, using a 40 mm (1.58 in.) diameter×200mm (8 in.) long silica gel column eluted with CHCl₃ and diethyl ether(Et₂ O) according to the following table (unless otherwise indicated,all ratios are v/v):

    ______________________________________                                        Solvent -(v/v)                                                                             Solvent volume (ml)                                                                          Fraction numbers                                  ______________________________________                                        CHCl.sub.3                                                                             100     500            01-22                                         CHCl.sub.3 /Et.sub.2 O                                                                 98/2    500            23-46                                         CHCl.sub.3 /Et.sub.2 O                                                                 95/5    1000           47-93                                         CHCl.sub.3 /Et.sub.2 O                                                                  90/10  500             94-118                                       ______________________________________                                    

A light yellow oily product (0.843 g; 59% theoretical yield) wasobtained by combining and evaporating fractions 81-105 (In theory, ayield of 1.43 g tetra-BBE-PET would be expected from 2.0 g of the crudeproduct placed on the column). The purified light yellow product wasconfirmed by analysis using a Beckman Acculab 2 infrared ("IR")spectrometer and a Varian FT-80 NMR spectrometer. The absence of a peakat 3500 cm-1 indicated the absence of hydroxyl functionality. Nuclearmagnetic resonance analysis (¹ H NMR (CDCl₃)) was consistent with thedesired product; aliphatic methylenes δ 3.6 (s, 8 H), benzylicmethylenes δ 8 4.5 (s, 8 H), and aromatics δ 7.15-7.65 (m, 36 H) versustetramethylsilane internal standard.

It is clear, therefore, that a reagent can be prepared having thedesired physical and chemical characteristics embodied in thisinvention. The reagent was used to couple various target molecules tosupport surfaces as described in EXAMPLES 3 through 13.

EXAMPLE 2 Preparation of Tetrakis (4-benzoylbenzoate ester) ofPentaerythritol [tetra-BBA-PET]

Pentaerythritol [Aldrich] (136 mg; 1 mmole), 4-benzoylbenzoyl chloride(1.0 g; 4.09 mmole; prepared by the reaction of thionyl chloride and4-benzoylbenzoic acid [Aldrich]), triethylamine [Aldrich] (696 ml; 5mmole), and chloroform (10 ml) were stirred overnight at roomtemperature. The reaction mixture was placed in ice cold hydrochloricacid (0.5M; 11 ml) and thoroughly mixed for 1 minute. The chloroformlayer was separated, dried over sodium sulfate, and evaporated, yieldingan orange residue (1.13 g). The residue was purified by flashchromatography using a 40 mm (1.57 in.) diameter by 180 mm (7 in.) longsilica gel column, which was eluted with chloroform/acetonitrile, 96:4(v/v). Seventy-two 13 ml fractions were collected. Fractions 37 to 61were combined and evaporated to give a white solid (322 mg; 33% oftheory). Analysis on a Varian FT-80 NMR spectrometer was consistent withthe desired product: ¹ H NMR (CDCl₃); aliphatic methylenes δ 5 4.7 (s,8H) and aromatics δ 7.15-8.10 (m, 36H) versus tetramethylsilane internalstandard.

Thus, it can be seen that a restrained multifunctional reagent can beprepared having ester groups as linkages. The reagent was used to modifypolymethylmethacrylate (PMMA) for application of polyvinylpyrrolidone(PVP) as demonstrated in EXAMPLE 14.

EXAMPLE 3 Surface Modification of Polymethylmethacrylate (PMMA) bySequential Application of tetra-BBE-PET and Polyvinylpyrrolidone (PVP)

A clear PMMA "coupon" (Rohm & Haas), 4 cm (1.57 in.)×2 cm (0.78 in.)×2mm (0.08 in.), was first wiped with an isopropyl alcohol (IPA) soakedtissue, after which one-half of the coupon was brush coated with a 0.1mg/ml solution of tetra-BBE-PET in IPA. After the coating had air-driedfor 5 minutes under ambient conditions, the entire coupon wasilluminated for 30 seconds, at a distance of 150 mm (6 in.) from a 100watt short arc mercury vapor bulb. After a rinse with excess IPA toremove any unbound tetra-BBE-PET, the entire coupon was then brushcoated with a 10 mg/ml solution of PVP (160,000 molecular weight; GAFChemical Corp.) in deionized (DI) water. After the PVP had air-dried(approx. 5 min.), the coupon was again illuminated for 30 seconds infront of the same light source. The coupon was then rubbed extensively(approx. 1 min.) under a flow of DI water to check the durability of thePVP coating.

After this rinse, the half of the coupon that was coated withtetra-BBE-PET remained noticeably more wettable and lubricious to thetouch than the half coated with PVP alone. The presence of the bound PVPon the tetra-BBE-PET coated half was verified by staining with a 0.35%solution of Congo Red (Sigma) in DI water.

EXAMPLE 4 Surface Modification of Polyethylene (PE) Tubing by SequentialApplication of tetra-BBE-PET and a Mixture of Photo-derivatized Polymers(1:1)

Pieces of PE tubing (25 cm (9.8 in.))×(1.0 mm outer diameter (0.04 in.))were first dip coated using a 0.1 mg/ml solution of tetra-BBE-PET inIPA. After the coating had air-dried (approx. 5 min.), the tetra-BBE-PETcoated tubing was illuminated for 3 minutes midway between two opposedELC-4000 lamps containing 400 watt metal halide/mercury vapor bulbsseparated by a distance of 91 cm (36 in.). After a rinse with excess IPAto remove any unbound tetra-BBE-PET, the tubing was then dipped andsubsequently withdrawn at a rate of 1.5 cm (0.59 in.)/sec from asolution containing 15 mg/ml photopoly[(acrylaniide)-co-(2-acrylamido-2-methylpropanesulfonic acid)]("photo-PA-AMPS ") and 15 mg/ml "photo-PVP" in water.

The photo-PA-AMPS was prepared by a copolymerization of acrylamide,2-acrylamide-2-methylpropanesulfonic acid ("AMPS"), andN-(3-aminopropyl)methacrylamide ("APMA"), followed byphotoderivatization of the polymer using 4-benzoylbenzoyl chloride underSchotten-Baumann conditions. The photo-PVP was prepared bycopolymerization of 1-vinyl-2-pyrrolidone and APMA, followed byphotoderivatization as described above. After the coating solution haddried (approx. 5 minutes at 55° C. (151 ° F.)), the tubing was againilluminated for 3 minutes.

Tubes coated with photo-PVP and photo-PA-AMPS alone have been shown toexhibit microscopic cracks, which can lead to flaking of the coating. Incontrast, the tubes that were first coated with tetra-BBE-PET, and thencoated with photo-PVP and photo-PA-AMPS, in the manner described above,showed little or no cracking.

EXAMPLE 5 Surface Modification of PE Tubing by Sequential Application oftetra-BBE-PET and a Mixture of Photo-PA-AMPS and Photo-PVP (2:1) (Wetillumination)

Pieces of PE tubing (25 cm, 9.8 in.)×(1.0 mm, O.D.,0.04 in.) were firstdip coated with tetra-BBE-PET using a 0.1 mg/ml solution of the reagentin IPA. The tetra-BBE-PET coated tubing was immediately illuminateduntil dry (approx. 3 minutes) midway between metal halide/mercury vaporbulbs in the manner described in EXAMPLE 4. After a rinse with excessIPA to remove any unbound tetra-BBE-PET, the tubing was then immersed ina solution containing 10 mg/ml photo-PA-AMPS and 5 mg/ml of photo-PVP in15% aqueous IPA, prepared in the manner described in Example 4, and thenwithdrawn at a rate of 1 cm (0.39 in.)/sec. The tubing was againilluminated until dry (approx. 3 minutes).

Tubes first coated with tetra-BBE-PET followed by photo-PVP andphoto-PA-AMPS showed little or no cracking when evaluated by lightmicroscopy, in contrast to previous experience with tubes similarlycoated although lacking tetra-BBE-PET.

EXAMPLE 6 Surface Modification of Silicone Tubing by SequentialApplication of tetra-BBE-PET and a Mixture of Photo-PA-AMPS andPhoto-PVP (2:1) (Wet Illumination)

Pieces of silicone tubing (38 cm, 15 in.)×(5 mm, O.D, 0.20 in.) (DowComing) were first dip coated using a 0.1 mg/ml solution oftetra-BBE-PET in IPA. The tetra-BBE-PET coated tubing was immediatelyilluminated until dry (approx. 3 minutes) midway between two opposedDymax PC-2 lamps containing 400 watt metal halide/mercury vapor bulbs,51 cm (20 in.) apart. After a rinse with IPA to remove any unboundtetra-BBE-PET, the tubing was then immersed into a solution containing10 mg/ml of photo-PA-AMPS and 5 mg/ml of photo-PVP in 15% aqueous IPA,prepared in the manner described in Example 4, and then withdrawn at arate of 1 cm (0.39 in.)/sec. The tubing was again illuminated until dry(approx. 3 minutes).

Extensive washing and rubbing of the surface with fingers indicated astrongly adherent layer of the lubricious photo-PA-AMPS/photo-PVP. Thepresence of the bound PVP on the surface was also verified by stainingwith a 0.35% solution of Congo Red in DI water.

The Congo Red stain on silicone tubing that was coated with onlyphoto-PVP and photo-PA-AMPS appeared spotty, indicative of areas wherethe coating was not bound to the surface and had therefore been rubbedoff. However, robes that were first coated with tetra-BBE-PET and thencoated with photo-PVP and photo-PA-AMPS appeared smoother and morecontiguous, indicating that the tetra-BBE-PET was useful in increasingthe tenacity and continuity of the PVP and PA coating.

EXAMPLE 7 Immobilization of PVP onto Polyvinylidene Difluoride (PVDF)Membranes Using tetra-BBE-PET

PVDF membranes, which are normally quite hydrophobic, were renderedhydrophilic by the treatment of the membranes with tetra-BBE-PET,followed by subsequent exposure to unmodified PVP.

PVDF Immobilon™-P Transfer Membranes (Millipore) were soaked for thirtyminutes in a solution of 0.2 mg/ml of tetra-BBE-PET in MeOH. Themembranes were removed from the tetra-BBE-PET solution and air-dried forfive minutes. The membranes were suspended midway between opposed DymaxPC-2 lamps (51 cm (20 in.) apart) and illuminated for two minutes. TheDymax lamps contained 400 watt metal halide/mercury vapor bulbs. Themembranes were washed three times with 100 ml of MeOH to remove unboundtetra-BBE-PET. After the final wash, the membranes were allowed toair-dry for five minutes.

The tetra-BBE-PET primed membranes were soaked for thirty minutes in PVP(Sigma, average MW of 360,000) solutions of varying concentration, (0 to40 mg/ml) in MeOH. The membranes were removed from the PVP solutions andallowed to air-dry for five minutes. The membranes were suspended midwaybetween opposed Dymax lamps 51 cm (20 in.) apart and illuminated for twominutes as described above. The membranes were washed for thirty minutesin 100 ml of MeOH with agitation. The MeOH wash was discarded and thewashing procedure repeated three times. After the final wash, themembranes were removed and air-dried for five minutes.

The hydrophilicity of the PVDF membranes was evaluated by droppingmembranes into a beaker filled with DI water and assessing their abilityto absorb water. PVDF membranes that were treated with tetra-BBE-PET andexposed to 10 mg/ml PVP or greater, absorbed water instantaneously whenplaced in a beaker filled with water. They became translucent and sankto the bottom of the beaker. Untreated PVDF membranes were completelynon-absorbent when placed in a beaker of water; they remained opaque andfloated on the surface of the water indefinitely.

EXAMPLE 8 Immobilization of Nitrocellulose on PVDF Membrane Usingtetra-BBE-PET

The incorporation of nitrocellulose (Hercules) onto PVDF membranes wasaccomplished by treatment of the membranes with tetra-BBE-PET followedby a subsequent exposure to unmodified nitrocellulose.

PVDF Immobilon™-P Transfer Membranes (Millipore) were soaked for thirtyminutes in a 0.2 mg/ml solution of tetra-BBE-PET in MeOH. The membraneswere removed from the tetra-BBE-PET solution and air-dried for 5minutes. The membranes were suspended midway between opposed Dymaxlamps, 51 cm (20 in.) apart, and illuminated for two minutes. The Dymaxlamps incorporated the same bulb as described in EXAMPLE 7. Themembranes were washed three times with 100 ml MeOH to remove unboundtetra-BBE-PET. After the final wash, the membranes were air-dried forfive minutes.

The tetra-BBE-PET primed membranes were soaked for thirty minutes in a40 mg/ml nitrocellulose solution. The nitrocellulose used was Type RSgrade 18-25, having a viscosity of 24 cps. The membranes were removedfrom the nitrocellulose solution and air-dried for five minutes. Themembranes were suspended between Dymax lamps and illuminated for twominutes as described above. The membranes were washed with agitation forthirty minutes in 100 ml of MeOH. The MeOH wash was discarded and thewashing procedure repeated three times. After the final wash themembranes were removed and allowed to air-dry for five minutes.

The protein binding characteristics of the primed membranes was comparedto those of native nitrocellulose (Schleicher - Schuell) and unprimedPVDF membranes by a simple dot-blot binding assay (adapted fromEasy-Titer ELIFA Septum Instructions, Pierce).

Bovine serum albumin CBSA", M.W.=66,000 Daltons) was dissolved inphosphate buffered saline (PBS) and serially diluted. Ten microliters ofeach dilution was pipetted into wells of the dot-blot manifold induplicate. A vacuum was applied to the manifold to yield a flow rate of14 ml/min. The presence of protein was determined with an enhancedcolloidal gold stain. (Collodial Gold Total Protein Stain - Catalog No.170-6527 and Gold Enhancement Kit - Catalog No. 170-6538, Bio-Rad).

Although all membranes tested detected 16 ng of protein (the limit ofthe assay), the signals generated on the hybrid membranes, as evaluatedby visual inspection, were more intense than those on eithernitrocellulose or unmodified PVDF membranes. This suggests that thehybrid membranes can provide a more sensitive assay matrix. Furthermore,the generation of stronger signals allows for a more definitiveevaluation of protein binding. The results showed 16 ng of protein onthe hybrid membranes gave a signal equivalent in intensity toapproximately 125 ng of protein on nitrocellulose. Unmodified PVDF gavean equivalent signal at 63 ng of protein.

Using a low molecular weight protein (aprotinin, MW=6,500 D), the hybridmembrane was as sensitive as nitrocellulose. Both detected 400 ng ofprotein with approximately equivalent intensity of signal. In contrast,the limit of sensitivity of PVDF was only 1.6 μg. In addition, theintensity of the signal generated by the 1.6 μg on PVDF was markedlylower than that on the hybrid membrane (approximately equivalent to thesignal generated by 400 ng on the hybrid membrane).

EXAMPLE 9 Co-Immobilization of Nitrocellulose on PVDF Membrane Usingtetra-BBE-PET

A coating solution was prepared by dissolving nitrocellulose (Type RSgrade 18-25 having a viscosity of 24 cps, Hercules Inc.) at 40 mg/ml andtetra-BBE-PET at 0.1 mg/ml in MeOH. PVDF Immobilon™-P transfer membranes(Millipore) were soaked in the coating solution for thirty minutes. Themembranes were removed and immediately suspended midway between opposedDymax lamps, 51 cm (20 in.) apart, and illuminated for two minutes. TheDymax lamps used were of the same specifications as previously mentioned(EXAMPLE 7). The membranes were washed three times with 100 ml MeOH withagitation. After the final wash the membranes were air-dried for fiveminutes.

A coating could be seen upon visual inspection of the membranes. Theprotein binding characteristics of the hybrid membranes were compared tothose of native nitrocellulose and PVDF membranes by a simple dot-blotbinding assay (adapted from Easy-Titer ELIFA Septum Instructions,Pierce).

BSA (MW=66,000 Daltons) was dissolved in PBS and serially diluted. Tenmicroliters of each dilution was pipetted into wells of the dot-blotmanifold in duplicate. A vacuum was applied to the manifold to yield aflow rate of 14 ml/min. The presence of protein was determined with anenhanced colloidal gold stain. (Collodial Gold Total Protein Stain -Catalog No. 170-6527 and Gold Enhancement Kit- Catalog No. 170-6538,Bio-Rad).

Similarly, all membranes tested detected 16 ng of protein (the limit ofthe assay) and the signals generated on the hybrid membranes were moreintense, as evaluated by visual inspection, than those on eithernitrocellulose or unmodified PVDF. Again this suggests that the hybridmembranes can provide a more sensitive assay matrix. Furthermore, thegeneration of stronger signals allows for a more definitive evaluationof protein binding. The results demonstrated that the signal of 16 ng ofprotein on the hybrid was more intense than the signal generated by 16ng on PVDF but not as intense as that generated by 31 ng on PVDF. Theintensity of the signal generated by 16 ng of protein on the hybridmembrane was approximately equivalent to that of 63 ng onnitrocellulose.

Again using a low molecular weight protein (aprotinin, MW=6,500 D), thehybrid membrane was as sensitive as nitrocellulose. Both detected 400 ngof protein with approximately equivalent intensity of signal. The limitof sensitivity of PVDF was only 1.6 μg. In addition, the intensity ofthe signal generated by 1.6 μg on PVDF was markedly lower than that onthe hybrid membrane (approximately equivalent to the signal generated by400 ng on the hybrid membrane).

EXAMPLE 10 Immobilization of Human Gamma Globulin (HGG) onto MicrotiterPlates Using tetra-BBE-PET

The covalent immobilization of HGG onto polystyrene microtiter plateswas accomplished by pretreatment of the plates with tetra-BBE-PETfollowed by a subsequent exposure to an HGG solution.

Ninety-six well breakable polystyrene microtiter plates (LabsystemsInc.) were prewashed using 200 μls MeOH per well. Solutions oftetra-BBE-PET were prepared in MeOH with concentrations ranging from 0to 0.5 mg/ml. The microtiter plates were divided into sections with eachsection receiving a different concentration of tetra-BBE-PET. Onehundred microliters of solution was pipetted into each well. Thesolutions were incubated in the plates for one hour at room temperature.After incubating, the tetra-BBE-PET solutions were removed from theplates by aspiration. The plates were air-dried for thirty minutes. Theplates were placed 48 cm (19 in.) beneath an ELC-4000 lamp andilluminated for two minutes. The ELC lamp uses a 400 watt metalhalide/mercury vapor bulb. The plates were washed with 200 μl MeOH perwell three times to remove unbound tetra-BBE-PET. The plates wereair-dried for thirty minutes.

The tetra-BBE-PET activated plates were subsequently exposed tosolutions of [³ H]-HGG in PBS Ph 7.2. Tritiated HGG was prepared by thereductive methylation of HGG with NaB[³ H]₄ and formaldehyde (Jentoft,N. and D. G. Dearborn, J. of Biol. Chem.254:4359 (1979). The solutionsof [³ H]-HGG ranged in concentration from 0 to 20 mg/ml. One hundredmicrotiters of the [³ H]-HGG solutions were added to each well. Thesolutions were incubated in the plates for 15 minutes at roomtemperature. The plates were placed 48 cm (19 in.) beneath an ELC lampand illuminated for four minutes. The solutions were aspirated from theplates and the plates washed six times with 200 μl of PBS containing0.05% TWEEN 20 per well.

The plates were broken apart and each well dissolved in two ml oftetrahydrofuran (THF). After dissolution of the wells was complete, 5 mlof liquid scintillation cocktail (Aquasol-2, Dupont) was added to eachvial and the vials read on a liquid scintillation analyzer (Packard 1900Calif.). From the total radioactivity found in each vial, the amount of[³ H]-HGG bound to each well can be calculated. The wells pre-treatedwith tetra-BBE-PET showed significantly greater amounts of [³ H]-HGGbound than did the untreated wells. This was particularly true at thehigher concentrations of protein. Using an [³ H]-HGG concentration of 20mg/ml, the tetra-BBE-PET pre-treated wells showed [³ H]-HGG bindingincreases of 37%, 52% and 56% over untreated wells at tetra-BBE-PETconcentration of 0.1, 0.2 and 0.5 mg/ml respectively.

EXAMPLE 11

    ______________________________________                                        Surface Modification of Polystyrene with Sequential                           Application of teta-BBE-PET and Collagen Type IV                              Group Treatment                                                               ______________________________________                                        1     tetra-BBE-PET, aspirated & dried, illuminated, rinsed,                        [.sup.3 H]collagen IV, aspirated & dried, illuminated.                  2     tetra-BBE-PET, aspirated & dried, [.sup.3 H]collagen IV,                      aspirated & dried, illuminated.                                         3     Illuminated surface, adsorbed [.sup.3 H]collagen IV,                          aspirated & dried.                                                      4     Adsorbed [.sup.3 H]collagen IV, aspirated & dried.                      5     Adsorbed [.sup.3 H]collagen IV, aspirated & dried, illuminated.         6     Photo[.sup.3 H]collgen IV, aspirated & dried, illuminated.              ______________________________________                                    

Polystyrene wells from a 96-well polystyrene break-away plate (DynatechImmulon I, inner diameter of 8.71 mm (0.343 inches) were coated withtetra-BBE-PET at 0.1 mg/ml in methanol (Groups 1 and 2). All wells werecovered with Parafilm® (American Can Company, Greenwich, Conn.) toprevent evaporation and were incubated for 1 hour at room temperature.After incubation, the tetra-BBE-PET solution was aspirated from thewells and the wells were air-dried for 5 minutes.

Wells from Group 1 were illuminated for 90 seconds, 20 cm (8 inches)from a Dymax PC-2 lamp. The Dymax lamp used contained a 400 watt metalhalide/mercury vapor bulb. Wells in Group 2 were not illuminated at thispoint. The wells from Group 1 were rinsed 3 times with methanol. Anotherset of uncoated polystyrene wells (Group 3) were illuminated asdescribed above. [³ H]Collagen IV, prepared from collagen IV (SigmaC-7521 ) by reductive methylation with NaB[³ H]₄ and formaldehyde (seeJentoft and Dearborn, above), was diluted to 0.1 mg/ml in PBS and addedto the wells of Groups 1,2,3,4, and 5.

Photoderivatized [³ H]collagen IV, prepared from collagen IV (SigmaC-7521) by reductive,methylation (see Example 10), followed byphotoderivatization using a process described in Example 1-3B of U.S.Pat. No. 4,973,493, was diluted to 0.1 mg/ml in PBS and added to thewells of Group 6. All wells were covered with parafilm to preventevaporation and were incubated for 1 hour at room temperature. Afterincubation, the reagents were aspirated from the wells and the wellswere air-dried for 10 minutes. Groups 1,2,4,5 and 6 were illuminated for90 seconds as described above.

All groups underwent three 1 hour washes, an overnight wash in 1% Tween20/PBS, followed by three 1 hour rinses in PBS. In preparation forscintillation counting, each well was dissolved in THF and mixed withscintillation cocktail. Finally, the number of disintegrations perminutes (DPM's) produced by each sample was measured using a Packard1900CA Tri-Carb Liquid Scintillation Analyzer. The number of DPMsproduced indicated the amount of [³ H]collagen IV or photo[³ H]collagenIV present on the surface of each piece.

The level of [³ H]collagen IV immobilized under each condition (Groups1-6) was as follows: Group 1=725 ng/cm² ; Group 2=789 ng/cm² ; Group3=81 ng/cm² ; Group 4=64 ng/cm² ; Group 5=331 ng/cm² ; and Group 6=715ng/cm². It has been calculated that a monolayer of collagen IV on asurface would require approximately 500 ng/cm². The levels of [³H]collagen IV immobilized in Groups 1 and 2 are sufficient to producesuch a monolayer of collagen IV.

Therefore, it is apparent that tetra-BBE-PET (Groups 1 and 2) increasesthe level of [³ H]collagen IV immobilization over levels achieved byadsorption to an unmodified surface (Group 4), by adsorption to anilluminated bare surface (Group 3), or by illumination of [³ H]collagenIV without tetra-BBE-PET present (Group 5). The use of photo [³ H]collagen IV (Group 6) results in approximately equivalent loadings asseen in Group 1 and 2; however, the collagen IV was photoderivatizedbefore use, thus the use of tetra-BBE-PET can eliminate the need forprior photoderivatization.

EXAMPLE 12 Immobilization of dDNA on Polystyrene Using Tetra-BBE-PET

Immobilization of double-stranded deoxyribonucleic acids ("dDNA") wasaccomplished by treatment of polystyrene microtiter plates withtetra-BBE-PET, followed by subsequent exposure of the modified surfacewith dDNA.

Radiolabeled DNA surface tenacity experiments were conducted to compare"raw" (i.e., untreated) polystyrene with the tetra-BBE-PET treatedsurface. To each of the 96 wells of standard medium-binding polystyrenemicrotiter strip plates (Costar, Inc.) was added 100 μl of 0.5 mg/mltetra-BBE-PET in methanol. The plate was placed 25 cm (10 in) from aDymax PC-2 lamp containing a 400 watt metal halide/mercury vapor bulbfor 1.5 minutes (untreated control wells were not so treated). Each wellwas then rinsed three times with 300 μl of methanol and the platesallowed to air-dry.

A volume of 100 μl ³² P-labelled Lambda DNA (50 kb dDNA bacteriophage,35 pg DNA per well, 24 nCi per well) in phosphate buffered saline, pH3.0, was added to each well of an untreated or (tetra-BBE-PET) treatedpolystyrene microtiter plate, incubated at ambient temperature for 1hour, then illuminated for 4 minutes at a distance of 25 cm (10 in) fromthe Dymax lamp in a refrigerated cabinet (one untreated plate and onetreated plate were not illuminated). The plates were then washed usingone of two protocols to remove non-covalently absorbed dDNA:

Pre-hybridization solution--50% formamide, 5×Denhardt's solution (from50×stock; 5 g Ficoll, 5 g PVP, 5 g BSA, 500 ml H₂ O), 5×"SSPE" (from20×stock: 174 g NaCl, 27.6 g NaH₂ PO₄ -H₂ O, 7.4 g EDTA, 1 liter H₂ O,pH 7.4), 0.1% SDS; 4×200 μl per well, followed by 1×300 μl for 60minutes at 40° C., or

Denaturing solution--0.4N NaOH, 0.25% SDS; 4×200 μl per well, followedby 1×300 μl for 15 minutes at 40° C.

Following the wash treatments, relative quantities of immobilized DNAwere determined by breaking apart each of the 96 wells, dissolving eachin 1.5 ml THF, adding 5 ml of scintillation cocktail (Aquasol-2,DuPont), and analyzing on a Packard 1900CA liquid scintillationanalyzer. The results are tabulated below.

    __________________________________________________________________________    AVERAGE DPM ± STANDARD DEVIATION OF IMMOBILIZED dDNA ON                    POLYSTYRENE MICROTITER PLATES                                                                                  tetra-BBE-PET                                                                           % INCREASE                         WASH             RAW WITH  tetra-BBE-                                                                          WITH      IN DNA                             TREATMENT  RAW   ILLUMINATION                                                                            PET   ILLUMINATION                                                                            DUE TO tetra-BBE-PET               __________________________________________________________________________    PRE-       352 ± 38                                                                         7152 ± 1165                                                                          585 ± 43                                                                         9202 ± 1442                                                                          22%                                HYBRIDIZATTON                                                                 DENATURING 65 ± 20                                                                          1651 ± 89                                                                            195 ± 18                                                                         2969 ± 98                                                                            44%                                __________________________________________________________________________

As can be seen, the immobilization of DNA using tetra-BBE-PET resultsin: a 26-fold increase over untreated polystyrene using thepre-hybridization solution wash method, and a 46-fold increase overuntreated polystyrene using the denaturing wash method. Since thisincrease may be due in part to the photoillumination of DNA ontountreated polystyrene, the increase due only to covalent immobilizationof DNA onto polystyrene using tetra-BBE-PET is indicated by a 22%increase in DNA using pre-hybridization wash and a 44% increase using adenaturing wash. These results clearly demonstrate that surfacemodification of polystyrene with tetra-BBE-PET followed by addition ofdDNA and photoillumination is an effective method of immobilizing DNA tothis support surface.

EXAMPLE 13 Immobilization of Horse Cytochrome c on PS usingTetra-BBE-PET

Horse Cytochrome c ("Cyt c") is a protein (MW approximately 12,400) thatis commonly used as a model system for immunochemical studies. Thestructure of Cyt c has been studied extensively, including its proteinsequence, tertiary structure, conformation of specific immunogenicepitopes, and structural changes that can occur when adsorbed to solidsurfaces. This globular protein is known to demonstrate limitedadherence to raw polystyrene, and is thought to be accompanied byprotein denaturation upon adsorption. (See, e.g., Jemmerson, R.,"Antigenicity and Native Structure of Globular Proteins: Low Frequencyof Peptide Reactive Antibodies", Proc. Nat. Acad. Sci. U.S.A., 84:9180,1987; Stevens, F. J., "Considerations of the Interpretation of theSpecificity of Monoclonal Antibodies Determined in Solid-PhaseImmunoassays," in Immunochemistry of Solid-Phase Immunoassay, J. E.Butler, ed., CRC Press, 233, 1991; and Jemmerson, R., "MultipleOverlapping Epitopes in the Three Antigenic Regions of Horse Cytochromec," J. Immunol. 138:213, 1987.)

Immobilization of Cyt c was accomplished by treating polystyrenemicrotiter plates with tetra-BBE-PET, followed by subsequent exposure ofthe modified surface with Cyt c.

Experiments were conducted using tritium-radiolabelled Cyt c to compareraw (i.e., untreated) polystyrene with tetra-BBE-PET treated surfaces.Standard medium-binding polystyrene microtiter strip plates (Costar,Inc.) were modified with 200 μl of 0.4 mg/ml tetra-BBE-PET in methanolin each of the 96 wells (untreated control wells were not coated). Avolume of 150 μl was immediately removed from each well, and the platewas placed 25 cm (10 in) from a Dymax PC-2 lamp as described in Example12 above for 2 minutes.

The contents of each well were then immediately aspirated using anautomated plate washer, and the plate was rinsed with 300 μl of IPA perwell. The tetra-BBE-PET treated plates were air-dried in a 15%humidity-controlled environment before further testing. A volume of 100μl ³ H-labelled horse Cyt c at 4.2 μg/ml (0.34 μCi per weld in 0.05Mcarbonate-bicarbonate buffer, pH 9.6, was added to untreated or treatedpolystyrene, incubated on an environmental shaker set to rotate at 200rpm at a temperature of 37° C. for 2 hours, then illuminated for 2minutes at a distance of 25 cm (10 in) from a Dymax lamp using a 400watt metal halide/mercury vapor bulb (one untreated plate and onetreated plate were not illuminated).

The plates were then aspirated, and washed as follows: four washes, eachwith 200 μl /well, of 50 mM Tris, 150 mM NaCl, and 0.05% (v/v) Tween-20,pH 7.5 ("TNT"), followed by an incubation with 200 μl TNT per well on anenvironmental shaker rotating at 200 rpm at 37° C. for 2 hours, andthree final washes, each with 300 μl TNT. Following the final wash, thepolystyrene wells were broken apart, dissolved in 1.5 ml THF, andcounted by liquid scintillation spectroscopy as described in Example 12above, using 5 ml scintillation fluor. The resulting disintegrations perminute ("dpm") were translated to average ng protein per well. Theresults of this experiment were:

    __________________________________________________________________________    CYTOCHROME c IMMOBILIZATION TO TETRA-BBE-PET                                  TREATED POLYSTYRENE                                                                            NOT ILLUMINATED                                                                           ILLUMINATED                                                       AFTER CYT c AFTER CYT c                                      PLATE TREATMENT  ADDITION    ADDITION                                         __________________________________________________________________________    RAW POLYSTYRENE  7.15 ± 2.58                                                                            79.67 ± 16.37                                 tetra-BBE-PET POLYSTYRENE                                                                      126.38 ± 12.4                                                                          173.33 ± 5.63                                 __________________________________________________________________________

As can be seen above, surface modification by covalent bonding of Cyt cto polystyrene via this photoreagent resulted in: a 24-fold increaserelative to adsorption to raw polystyrene; a 2-fold increase relative toUV light-potentiated adsorption to raw polystyrene, and, a 1.4-foldincrease relative to tetra-BBE-PET treated polystyrene withoutphotoimmobilization (illumination) of the Cyt c to the surface. Theseresults clearly demonstrate that the tetra-BBE-PET treated polystyrenesurface is useful for covalent immobilization of proteins to this solidsupport matrix.

EXAMPLE 14 Surface Modification of Polyethylmethacrylate (PMMA) bySequential Application of tetra-BBA-PET and Polyvinlypyrolidone (PVP)

A clear PMMA coupon (Rohm & Haas), 4 cm (1.57 in.×2 cm (0.78 in.)×2 mm(0.08 in.) was wiped with an IPA soaked tissue, after which one-half ofthe coupon was brushed with a 0.08 mg/ml solution of tetra-BBA-PET inmethanol. After the coating had air-dried for 5 minutes under normallaboratory conditions, the entire coupon was illuminated for 30 seconds,150 mm (6 in.) from a 100 watt short arc mercury vapor bulb. After arinse with excess IPA to remove unbound tetra-BBA-PET, the entire couponwas then brush coated with 10 mg/ml of PVP (160,000 molecular weight no.ave. value, GAF Chemical Corp.) in DI water. After the PVP had air-dried(approximately 5 minutes), the coupon was again illuminated for 30seconds, 150 mm (6 inches) from the same light source and in the samemanner. The coupon was then rubbed extensively between fingers(approximately 1 minute) under a flow of DI water to check thedurability of the PVP coating.

After this rinse, the half coated with tetra-BBA-PET remained noticeablymore wettable and lubricious to the touch than the half of the couponthat was coated with PVP alone. The presence of the bound PVP on thetetra-BBA-PET coated half was verified by staining with a 0.35% solutionof Congo Red (Sigma) in DI water.

We claim:
 1. A restrained, multifunctional reagent selected from the group consisting of the tetrakis (4-benzoylbenzyl ether) and the tetrakis (4-benzoylbenzoate ester) of pentaerthyritol.
 2. A restrained, multifunctional reagent according to claim 1 wherein the reagent comprises the tetrakis (4-benzoylbenzyl ether) of pentaerythritol.
 3. A restrained, multifunctional reagent according to claim 2 wherein a plurality of the 4-benzoylbenzyl ether groups have been activated in order to attach them to a surface.
 4. A restrained, multifunctional reagent according to claim 2 wherein a plurality of the 4-benzoylbenzyl ether groups have been activated in order to attach them to a target molecule.
 5. A restrained, multifunctional reagent according to claim 2 wherein a plurality of the 4-benzoylbenzyl ether groups have been activated in order to attach them to a target molecule and a different plurality have been activated in order to attach them to a surface.
 6. A restrained, multifunctional reagent according to claim 4 wherein the target molecule is selected from the group consisting of synthetic polymers, carbohydrates, proteins, lipids, nucleic acids, drugs, vitamins, and cofactors.
 7. A restrained, multifunctional reagent according to claim 3 wherein the surface provides abstractable hydrogens suitable for covalent bonding with the activated group.
 8. A restrained, multifunctional reagent according to claim 7 wherein the surface is provided by a support selected from the group consisting of polyolefins, polystyrenes, poly(methyl)methacrylates, polyacrylonitriles, poly(vinylacetates), poly (vinyl alcohols), chlorine-containing polymers such as poly(vinyl) chloride, polyoxymethylenes, polycarbonates, polyamides, polyimides, polyurethanes, phenolics, amino-epoxy resins, polyesters, cellulose derivatives, silicones, and rubber-like plastics.
 9. A restrained, multifunctional reagent according to claim 1 wherein the reagent comprises the tetrakis (4-benzoylbenzoate ester) of pentaerythritol.
 10. A restrained, multifunctional reagent according to claim 9 wherein a plurality of the 4-benzoylbenzoate ester groups have been activated in order to attach them to a surface.
 11. A restrained, multifunctional reagent according to claim 9 wherein a plurality of the 4-benzoylbenzoate ester groups have been activated in order to attach them to a target molecule.
 12. A restrained, multifunctional reagent according to claim 9 wherein a plurality of the 4-benzoylbenzoate ester groups have been activated in order to attach them to a target molecule and a different plurality have been activated in order to attach them to a surface.
 13. A restrained, multifunctional reagent according to claim 11 wherein the target molecule is selected from the group consisting of synthetic polymers, carbohydrates, proteins, lipids, nucleic acids, drugs, vitamins, and cofactors.
 14. A restrained, multifunctional reagent according to claim 10 wherein the surface provides abstractable hydrogens suitable for covalent bonding with the activated group.
 15. A restrained, multifunctional reagent according to claim 14 wherein the surface is provided by a support selected from the group consisting of polyolefins, polystyrenes, poly(methyl)methacrylates, polyacrylonitriles, poly(vinylacetates), poly (vinyl alcohols), chlorine-containing polymers such as poly(vinyl) chloride, polyoxymethylenes, polycarbonates, polyamides, polyimides, polyurethanes, phenolics, amino-epoxy resins, polyesters, cellulose derivatives, silicones, and rubber-like plastics.
 16. A restrained, multifunctional reagent comprising an acylated derivative of tetraphenylmethane.
 17. A restrained, multifunctional reagent according to claim 16 wherein the reagent a plurality of the acyl groups have been activated in order to attach them to a surface.
 18. A restrained, multifunctional reagent according to claim 16 wherein a plurality of the acyl groups have been activated in order to attach them to a target molecule.
 19. A restrained, multifunctional reagent according to claim 16 wherein a plurality of the acyl groups have been activated in order to attach them to a target molecule and a different plurality have been activated in order to attach them to a surface.
 20. A restrained, multifunctional reagent according to claim 19 wherein the target molecule is selected from the group consisting of synthetic polymers, carbohydrates, proteins, lipids, nucleic acids, drugs, vitamins, and cofactors, and the surface is provided by a support selected from the group consisting of polyolefins, polystyrenes, poly(methyl)methacrylates, polyacrylonitriles, poly(vinylacetates), poly (vinyl alcohols), chlorine-containing polymers such as poly(vinyl) chloride, polyoxymethylenes, polycarbonates, polyamides, polyimides, polyurethanes, phenolics, amino-epoxy resins, polyesters, cellulose derivatives, silicones, and rubber-like plastics. 